Xinmailong Attenuates Doxorubicin-Induced Lysosomal Dysfunction and Oxidative Stress in H9c2 Cells via HO-1.

The clinical use of doxorubicin (DOX) is limited by its cardiotoxicity, which is closely associated with oxidative stress. Xinmailong (XML) is a bioactive peptide extracted from American cockroaches, which has been mainly applied to treat chronic heart failure in China. Our previous study showed that XML attenuates DOX-induced oxidative stress. However, the mechanism of XML in DOX-induced cardiotoxicity remains unclear. Heme oxygenase-1 (HO-1), an enzyme that is ubiquitously expressed in all cell types, has been found to take antioxidant effects in many cardiovascular diseases, and its expression is protectively upregulated under DOX treatment. Lysosome and autophagy are closely involved in oxidative stress as well. It is still unknown whether XML could attenuate doxorubicin-induced lysosomal dysfunction and oxidative stress in H9c2 cells via HO-1. Thus, this study was aimed at investigating the involvement of HO-1-mediated lysosomal function and autophagy flux in DOX-induced oxidative stress and cardiotoxicity in H9c2 cells. Our results showed that XML treatment markedly increased cell proliferation and SOD activity, improved lysosomal function, and ameliorated autophagy flux block in DOX-treated H9c2 cells. Furthermore, XML significantly increased HO-1 expression following DOX treatment. Importantly, HO-1-specific inhibitor (Znpp) or HO-1 siRNA could significantly attenuate the protective effects of XML against DOX-induced cell injury, oxidative stress, lysosomal dysfunction, and autophagy flux block. These results suggest that XML protects against DOX-induced cardiotoxicity through HO-1-mediated recovery of lysosomal function and autophagy flux and decreases oxidative stress, providing a novel mechanism responsible for the protection of XML against DOX-induced cardiomyopathy.

Immunostimulatory and anti-allergic potential of novel heterotrimeric lectin from seeds of Zizyphus mauritiana Lam.

Zizyphus mauritiana Lam. seeds (ZMS) have been used medicinally as sedative or hypnotic drugs in most of Asian countries. ZMS has significant benefits to the human health. Therefore, we have evaluated immunomodulatory effect of lectin extracted from these ZMSL in both in vitro and in vivo study. Anaphylaxis is a severe life-threatening allergic reaction and Arthus reaction is deposition of immune complex and complement system activation, so we hypothesized that if ZMSL can protect these severe allergic diseases. We have studied the effect of ZMSL on macrophages and Wistar albino rats and confirmed its protective effect against anaphylaxis and Arthus reaction. Results of this study suggest ZMSL have immunostimulatory and antiallergic activity.

EGCG exerts its protective effect by mitigating the release of lysosomal enzymes in aged rat liver on exposure to high cholesterol diet.

The aim is to test the hypothesis whether the cholesterol loaded lysosomes are capable of mediating lysosomal membrane permeabilization (LMP) during aging and to study the efficacy of epigallocatechin-3-gallate (EGCG) in preserving the lysosomal membrane stability. Aged rats were fed with high cholesterol diet (HCD) and treated with EGCG orally. Serum and tissue lipid status, cholesterol levels in lysosomal fraction, activities of lysosomal enzymes in lysosomal, and cytosolic fractions were measured. Transmission electron microscopic studies (TEM), oil red "O" (ORO) staining, and immunohistochemical analysis of oxidized low density lipoprotein (OxLDL) were carried out. Significant increase in serum, tissue lipid profile, and lysosomal cholesterol levels were observed in aged HCD-fed rats with a concomitant decrease in high density lipoprotein (HDL) levels. We also observed a significant increase in lipid accumulation in hepatocytes of aged HCD-fed rats by TEM, ORO, and immunohistochemical staining. Upon treatment with EGCG to aged HCD-fed animals, we found augmented levels of HDL with a concomitant decrease in lysosomal cholesterol levels and other lipoproteins. TEM studies and immunohistochemistry of OxLDL also showed a marked reduction in lipid deposition of hepatocytes. Thus, EGCG has preserved the lysosomal membrane stability in HCD stressed aged rats. SIGNIFICANCE OF THE STUDY: The research article is focused mainly on the effect of EGCG and its capability on mitigating the release of lysosomal enzymes in aged animals fed with HCD. The study signifies the cellular function of the organelle lysosome following administration of aged rats with HCD, which would make the readers to understand the action of EGCG and the interrelationship of both cholesterol and activity of lysosomes when cholesterol is loaded.

Lycopene improves methotrexate-induced functional alterations of the Madin-Darby kidney cells in a concentration-dependent manner.

Lycopene is one of the most potent antioxidants among carotenoids due to its ability to quench singlet oxygen and react with free radicals to reduce DNA damage. Methotrexate is widely used in the treatment of several types of cancers and autoimmune diseases. One of the most common side effects of a high-dose of methotrexate is kidney injury. In this study, we evaluated effects of lycopene on the Madin-Darby canine kidney cells (MDCK) treated with methotrexate through the estimation of their mitochondrial and lysosomal functions ((4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide reduction assay and neutral red uptake assay) and changes in cell oxidative status (determination of advanced oxidized proteins concentrations and reduced glutathione levels) and lysosomal enzymes activity (ß-N-acetyl glucosaminidase activity). Results of our study showed that lycopene applied in high concentration caused significant impairment of the MDCK function leading to cell death. Contrarily, in relatively low concentrations lycopene moderately ameliorated methotrexate-induced MDCK cell death estimated by both biochemical and microscopic analyses. It also prevented a significant decline in the MDCK cell lysosomal function estimated by neutral red accumulation ability and activity of the lysosomal enzyme ß-N-acetyl glucosaminidase.

Activity of Lysosomal Enzymes During Protein Malnutrition and Progesterone Supplementation in the Mouse.

This study investigated the effects of protein malnutrition and progesterone supplementation on the activities of a spectrum of lysosomal enzymes in tissue fragments of mouse liver and kidney. The working hypothesis was that the known anti-stress action of progesterone could have to do with the inhibition of lysosomes which are engaged in apoptotic and oxidative stress-induced responses. The study investigated the effects of exogenous progesterone in chronically (3 weeks) protein-malnourished (10% protein) mice on the activities of lysosomal hydrolases in liver and kidney tissues. Progesterone was injected intraperitoneally in a dose of 2 µg/g body mass dissolved in a vehicle volume of 10 µL/g body mass during the final 3 days of exposure to either low 10% or standard 16% protein content in the chow. After euthanizing the animals, tissue fragments of liver and kidney assayed for the content of lysosomal enzymes. The results demonstrated the stimulating effect of protein malnutrition on lysosomal activities. We further found, contrary to our hypothesis, that progesterone supplementation during both standard and low-protein conditions enhanced lysosomal activities, particularly acting in concert with protein malnutrition in kidney tissue. The effects were selective concerning both lysosomal enzymes and tissues and of highly variable magnitude. Nonetheless, we believe we have shown that progesterone assists protein malnutrition in stimulation of lysosomal enzymes, which suggests the possibility of the hormone's engagement in cleansing the cellular milieu in disorders consisting of accumulation of toxic molecules.

The role of lysosomal cysteine cathepsins in NLRP3 inflammasome activation.

Lysosomal cysteine cathepsins are a family of proteases that are involved in a myriad of cellular processes from proteolytic degradation in the lysosome to bone resorption. These proteins mature following the cleavage of a pro-domain in the lysosome to become either exo- or endo-peptidases. The cathepsins B, C, L, S and Z have been implicated in NLRP3 inflammasome activation following their activation with ATP, monosodium urate, silica crystals, or bacterial components, among others. These five cathepsins have both compensatory and independent functions in NLRP3 inflammasome activation. There is much evidence in the literature to support the release of cathepsin B following lysosomal membrane degradation which leads to NLRP3 inflammasome activation. This is likely due to a hitherto unidentified role of this protein in the cytoplasm, although other interactions with autophagy proteins and within lysosomes have been proposed. Cathepsin C is involved in the processing of neutrophil IL-1ß through processing of upstream proteases. Cathepsin Z is non-redundantly required for NLRP3 inflammasome activation following nigericin, ATP and monosodium urate activation. Lysosomal cysteine cathepsins are members of a diverse and complementary family, and likely share both overlapping and independent functions in NLRP3 inflammasome activation.

Anti-inflammatory Effect of Total Saponin Fraction from Dioscorea nipponica Makino on Gouty Arthritis and Its Influence on NALP3 Inflammasome.

OBJECTIVE: To investigate the mechanism of Chinese herbal medicine Dioscorea nipponica for the treatment of monosodium urate crystals-induced gouty arthritis (GA) in rats. METHODS: Sixty male Wistar rats were divided into 6 groups: normal, model, indomethacin and three total saponin (900, 300 and 100 mg/kg) groups. The liver, kidney and serum levels of lysosomal enzymes, antioxidant capacities, and inflammatory factors were measured. In addition, the mRNA and protein levels of the NALP3 inflammasome components in the mononuclear cells of rats' peripheral blood were analyzed using real-time polymerase chain reaction and Western blotting methods, respectively. RESULTS: Total saponins groups could reduce the activities of ß-galactosidase, ß-N acetyl glucosamine enzyme, ß-glucuronidase, acid phosphatase, and malonaldehyde as well as the contents of TNF-α, IL-1ß and IL-8 (all P<0.05). They could also increase the activities of glutathione peroxidase and total superoxide dismutase (both P<0.05). Further studies showed that total saponins groups of high, middle and low doses could all increase the mRNA and protein levels of caspase-1, adapter apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) and NALP3 in the mononuclear cells of peripheral blood (all P<0.05). CONCLUSION: Dioscorea nipponica may treat GA by regulating lysosomal enzymes, antioxidant capacities and the NALP3 inflammasome.

Chloroquine aggravates the arsenic trioxide (As2O3)-induced apoptosis of acute promyelocytic leukemia NB4 cells via inhibiting lysosomal degradation in vitro.

OBJECTIVE: Acute promyelocytic leukemia (APL) is a distinct subtype of acute myeloid leukemia (AML), as standing out for its distinguished sensitivity to all-trans retinoic acid and arsenic trioxide (ATO, As2O3). The As2O3-mediated degradation of PML-RARA (promyelocytic leukemia-retinoic acid receptor-α) oncoprotein via the proteasome pathway appears to be critical for such distinguished sensitivity. MATERIALS AND METHODS: The present study was to evaluate the influence by chloroquine (CQ), an inhibitor to the release of lysosomal enzymes, on the sensitivity of APL cells to As2O3. APL-derived NB4 cell line was treated with As2O3 or/and CQ in vitro. Then, the cell viability, the induction of apoptosis, and autophagy were examined with MTT assay, with TUNEL staining or with enhanced green fluorescence protein (EGFP)-light Chain 3 (LC3) reporter. The apoptosis- or autophagy-associated proteins were quantified with Western blotting assay. RESULTS: Our results demonstrated that the As2O3 treatment promoted either apoptosis or autophagy in APL NB4 cells and upregulated both apoptosis- and autophagy-associated proteins. However, additional CQ treatment deteriorated the As2O3-induced NB4 cell apoptosis, whereas aggravated the As2O3-induced accumulation of acidic vesicular organelles (AVOs) and blocked the lysosomal degradation in NB4 cells. CONCLUSIONS: Chloroquine aggravates the arsenic trioxide-induced apoptosis of APL NB4 cells via inhibiting lysosomal degradation in vitro. It implies that chloroquine might be adjuvant to sensitize APL cells to arsenic trioxide.

Whey Protein Concentrate WPC-80 Intensifies Glycoconjugate Catabolism and Induces Oxidative Stress in the Liver of Rats.

The aim of this study was to evaluate the effect of whey protein concentrate (WPC-80) on glycoconjugate catabolism, selected markers of oxidative stress and liver inflammation. The experiment was conducted on male Wistar rats (n = 63). The animals from the study group were administered WPC-80 at a dose of 0.3 or 0.5 g/kg body weight for 7, 14 or 21 days, while rats from the control group received only 0.9% NaCl. In liver homogenates, we assayed the activity of N-acetyl-ß-D-hexosaminidase (HEX), ß-glucuronidase (GLU), ß-galactosidase (GAL), α-mannosidase (MAN), α-fucosidase (FUC), as well as the level of reduced glutathione (GSH), malondialdehyde (MDA), interleukin-1ß (IL-1ß) and transforming growth factor-ß1 (TGF-ß1). A significantly higher activity of HEX, GLU, MAN and FUC were found in the livers of rats receiving WPC-80 compared to controls. Serum ALT and AST were significantly higher in the animals supplemented with WPC-80 at a dose of 0.5 g/kg body weight for 21 days. In the same group of animals, enhanced level of GSH, MDA, IL-1ß and TGF-ß1 were also observed. WPC-80 is responsible for intensive remodelling of liver tissue and induction of oxidative stress especially at a dose of 0.5 g/kg body weight.

Effect of Metabolic Stress on Lysosomal Proteolysis in the Liver and Brain of Rats Receiving Q10-Enriched Ration.

Activation of lysosomal proteolysis and increased coenzyme Q10 level were revealed in the liver and brain of rats with individual typological features under conditions of food deprivation. Changes in cathepsin B activity in these tissues were different in behaviorally active and passive animals. We observed indirect modulating effect of coenzyme Q10 in the regulation of activity of lysosomal endopeptidases in the brain of animals with different behavior during adaptation to metabolic stress.

N-Butyl-l-deoxynojirimycin (l-NBDNJ): Synthesis of an Allosteric Enhancer of α-Glucosidase Activity for the Treatment of Pompe Disease.

The highly stereocontrolled de novo synthesis of l-NBDNJ (the unnatural enantiomer of the iminosugar drug Miglustat) and a preliminary evaluation of its chaperoning potential are herein reported. l-NBDNJ is able to enhance lysosomal α-glucosidase levels in Pompe disease fibroblasts, either when administered singularly or when coincubated with the recombinant human α-glucosidase. In addition, differently from its d-enantiomer, l-NBDNJ does not act as a glycosidase inhibitor.

Transcriptome profiling reveals the immune response of goose T cells under selenium stimuli.

The goose is an economically important poultry species and a principal natural host of avian viruses. This study aimed to determine the effects of selenium on the immune response of geese. Under selenium stimulation, gene expression profiling was investigated using transcriptome sequencing. The selenoproteins were promoted by selenium stimulation, while the heat shock proteins, interleukin and interferons were mainly down-regulated. After comparison, 2228 differentially expressed genes were primarily involved in immune and environmental response, and infectious disease and genetic information processing related pathways were identified. Specifically, the enzymes of the lysosomes which acted as a safeguard in preventing pathogens were mostly up-regulated and six randomly selected differentially expressed genes were validated by quantitative polymerase chain reaction. In addition, the most proportional increased transcription factor family basic helix-loop-helix (bHLH) located in the 5' flank of selenoprotein P-like protein for selenium metabolism was identified by response to the selenium stimulation in this study. These analyses show that selenium can promote immune function by activating selenoproteins, transcript factors and lysosome pathway related genes, while weakening cytokine content genes in geese.

Molecular characterization of aspartylglucosaminidase, a lysosomal hydrolase upregulated during strobilation in the moon jellyfish, Aurelia aurita.

The life cycle of the moon jellyfish, Aurelia aurita, alternates between a benthic asexual polyp stage and a planktonic sexual medusa (jellyfish) stage. Transition from polyp to medusa is called strobilation. To investigate the molecular mechanisms of strobilation, we screened for genes that are upregulated during strobilation using the differential display method and we identified aspartylglucosaminidase (AGA), which encodes a lysosomal hydrolase. Similar to AGAs from other species, Aurelia AGA possessed an N-terminal signal peptide and potential N-glycosylation sites. The genomic region of Aurelia AGA was approximately 9.8 kb in length and contained 12 exons and 11 introns. Quantitative RT-PCR analysis revealed that AGA expression increased during strobilation, and was then decreased in medusae. To inhibit AGA function, we administered the lysosomal acidification inhibitors, chloroquine or bafilomycin A1, to animals during strobilation. Both inhibitors disturbed medusa morphogenesis at the oral end, suggesting involvement of lysosomal hydrolases in strobilation.

Duvoglustat HCl Increases Systemic and Tissue Exposure of Active Acid α-Glucosidase in Pompe Patients Co-administered with Alglucosidase α.

Duvoglustat HCl (AT2220, 1-deoxynojirimycin) is an investigational pharmacological chaperone for the treatment of acid α-glucosidase (GAA) deficiency, which leads to the lysosomal storage disorder Pompe disease, which is characterized by progressive accumulation of lysosomal glycogen primarily in heart and skeletal muscles. The current standard of care is enzyme replacement therapy with recombinant human GAA (alglucosidase alfa [AA], Genzyme). Based on preclinical data, oral co-administration of duvoglustat HCl with AA increases exposure of active levels in plasma and skeletal muscles, leading to greater substrate reduction in muscle. This phase 2a study consisted of an open-label, fixed-treatment sequence that evaluated the effect of single oral doses of 50 mg, 100 mg, 250 mg, or 600 mg duvoglustat HCl on the pharmacokinetics and tissue levels of intravenously infused AA (20 mg/kg) in Pompe patients. AA alone resulted in increases in total GAA activity and protein in plasma compared to baseline. Following co-administration with duvoglustat HCl, total GAA activity and protein in plasma were further increased 1.2- to 2.8-fold compared to AA alone in all 25 Pompe patients; importantly, muscle GAA activity was increased for all co-administration treatments from day 3 biopsy specimens. No duvoglustat-related adverse events or drug-related tolerability issues were identified.

Dietary gamma oryzanol plays a significant role in the anti-inflammatory activity of rice bran oil by decreasing pro-inflammatory mediators secreted by peritoneal macrophages of rats.

Ricebran oil (RBO) is promoted as heart friendly oil because of its ability to maintain serum lipids at desirable levels. Inflammation also plays an important role on cardiovascular health. The role of minor constituents present in unsaponifiable fraction (UF) of RBO on inflammatory markers is not well understood. To evaluate this, we have taken RBO with UF (RBO-N), RBO stripped of UF (RBO-MCR) and RBO-MCR supplemented with UF from RBO (UFRBO) or Gamma-Oryzanol (γ-ORY) were added in AIN-93 diets which was then fed to Wistar rats for a period of 60 days. Groundnut oil with UF (GNO-N), UF removed GNO (GNO-MCR) and GNO-MCR supplemented with UF from RBO or γ-ORY was also used for comparison. The peritoneal macrophages from the rats were activated and pro-inflammatory mediators such as Reactive Oxygen Species (ROS), eicosanoids, cytokines, hydrolytic enzymes of lysosomal origin were monitored. The results indicated that UF of RBO and γ-ORY supplemented in the dietary oils play a significant role in reducing the secretion of pro-inflammatory mediators by macrophages. Hence γ-ORY in RBO significantly contributed to the anti-inflammatory properties of RBO.

Revisiting the neuronal localization and trafficking of CLN3 in juvenile neuronal ceroid lipofuscinosis.

Juvenile neuronal ceroid lipofuscinosis, the most common neurodegenerative disease affecting children, is caused by mutations of the CLN3 gene encoding CLN3, a transmembrane protein with so far undefined function. The embryonic expression of the gene has not been studied in detail before. Moreover, the protein CLN3 was mostly localized on the subcellular level to lysosomes but the exclusiveness is still under debate. Here, we analyze the expression pattern of murine CLN3 at different developmental stages by in situ hybridizations. We observe expression maxima in the developing thalamus and cerebral cortex and outside of the central nervous system in the gastrointestinal tract and other peripheral organs. In differentiated primary neurons, the protein CLN3 shows mainly a somatodendritic localization. In primary neurons, we thoroughly revisit the subcellular localization of CLN3 and find a predominant localization in late endosomal-lysosomal compartments. Moreover, we expressed the major mutant form of CLN3 - CLN3deltaExon7/8 - in neurons and demonstrate that it is retained in the endoplasmatic reticulum. Time-lapse microscopy analysis of neurons revealed co-trafficking of CLN3 with the late endosomal marker Rab7, but not with the early endosomal marker Rab5. Furthermore, a constitutive active mutant of Rab7 traps CLN3 in enlarged endosomes. Our subcellular localization study in neurons refines the localization and subcellular targeting of CLN3 to late endosomal-lysosomal compartments and provides information on the velocity of CLN3 in living neurons which has not been investigated before.

Immunomodulatory and cellular antioxidant activities of pure compounds from Teucrium ramosissimum Desf.

Evaluation of the immunomodulatory activity of plant compounds is an interesting and growing area of research. Teucrium ramosissimum Desf. is a native and endemic medicinal plant from the South of Tunisia traditionally used for the treatment of many diseases. The anti-inflammatory activity of apigenin-7-glucoside, genkwanin, and naringenin isolated from T. ramosissimum were assayed. The phagocytic activities of macrophage and lymphocyte proliferation were investigated in the absence and presence of mitogens (lipopolysaccharide [LPS] or lectin). Depending on the concentrations, the compounds affect macrophage functions by modulating their lysosomal enzyme activity and nitric oxide (NO) release. The tested compounds enhance significantly splenocyte proliferation, either with or without mitogen stimulation. In studies to assess any potential effects of apigenin-7-glucoside, genkwanin, and naringenin on innate immunity, the results showed that these compounds significantly enhanced the killing activity of natural killer (NK) cells and cytotoxic activity of the T lymphocyte (CTL) isolated from splenocytes. These results suggest that T. ramosissimum compounds such as apigenin-7-glucoside, genkwanin, and naringenin may be potentially useful for modulating immune cell functions in physiological and pathological conditions.

Práticas de cuidado ao idoso indígena - atuação dos profissionais de saúde / The elderly care practices of indigenous-performance of health / Las prácticas de cuidado de ancianos indígenas de rendimiento de la salud

Este estudo objetivou compreender as práticas de cuidado dos profissionais de saúde que assistem os idosos Kaingang. Estudo qualitativo, apoiado na etnografia, realizado com dez profissionais à que atuam na atenção primária saúde da Terra Indígena Faxinal, Paraná, Brasil. Os dados foram coletados no período de novembro de 2010 a fevereiro de 2012 por meio da observação participante e entrevistas, e, analisados à luz da Teoria Transcultural do Cuidado. Identificaram-se como práticas de cuidado a medicação e imunização, bem como, cuidados da medicina tradicional. Para realização destes cuidados, os profissionais dispunham de estratégias que proporcionavam manutenção dos idosos na assistência. Conclui-se que valores culturais e científicos necessitam integrar a assistência para melhoria da saúde dos idosos indígenas.

This research aims to understand the care practices of health professionals who assist the elderly Kaingang. It is a qualitative study, supported in ethnography, conducted by ten professionals working in primary health care in the indigenous land of Faxinal, Paraná, Brazil. The data was collected from November 2010 to February 2012 by participant observation and interviews, and analyzed based on the Transcultural Care Theory. Was identified the preoccupation of the carers practices with the medication and immunization, as well as traditional medical care. To achieve these, care professionals had strategies that implemented maintenance of older people in care. We conclude that cultural values and integrate scientific need assistance to improve the health of elderly indigenous.

Este estudio tuvo como objetivo entender las prácticas de cuidado de los profesionales de la salud que asisten a los ancianos Kaingang. Estudio cualitativo, apoyado en la etnografía, llevado a cabo con diez profesionales que trabajan en la atención primaria de la salud de la tierra indígena de Faxinal, Paraná, Brasil. Los datos fueron recogidos a partir de noviembre 2010 a febrero 2012 a través de la observación participante y las entrevistas, y analizado con base en la Teoría del Cuidado Transcultural. Se identificaron las prácticas de atención médica y imunizacion,el cuidado de la medicina, así tradicional. Para lograrlo, los profesionales tenían estrategias que proporcionaban el mantenimiento de las personas mayores en su atención. Se concluye que los valores culturales y científicos necesitan ayuda para mejorar la salud de los ancianos indígenas.

Suppression of mTORC1 activation in acid-α-glucosidase-deficient cells and mice is ameliorated by leucine supplementation.

Pompe disease is due to a deficiency in acid-α-glucosidase (GAA) and results in debilitating skeletal muscle wasting, characterized by the accumulation of glycogen and autophagic vesicles. Given the role of lysosomes as a platform for mTORC1 activation, we examined mTORC1 activity in models of Pompe disease. GAA-knockdown C2C12 myoblasts and GAA-deficient human skin fibroblasts of infantile Pompe patients were found to have decreased mTORC1 activation. Treatment with the cell-permeable leucine analog L-leucyl-L-leucine methyl ester restored mTORC1 activation. In vivo, Pompe mice also displayed reduced basal and leucine-stimulated mTORC1 activation in skeletal muscle, whereas treatment with a combination of insulin and leucine normalized mTORC1 activation. Chronic leucine feeding restored basal and leucine-stimulated mTORC1 activation, while partially protecting Pompe mice from developing kyphosis and the decline in muscle mass. Leucine-treated Pompe mice showed increased spontaneous activity and running capacity, with reduced muscle protein breakdown and glycogen accumulation. Together, these data demonstrate that GAA deficiency results in reduced mTORC1 activation that is partly responsible for the skeletal muscle wasting phenotype. Moreover, mTORC1 stimulation by dietary leucine supplementation prevented some of the detrimental skeletal muscle dysfunction that occurs in the Pompe disease mouse model.

Dying neurons in thalamus of asphyxiated term newborns and rats are autophagic.

OBJECTIVE: Neonatal hypoxic-ischemic encephalopathy (HIE) still carries a high burden by its mortality and long-term neurological morbidity in survivors. Apart from hypothermia, there is no acknowledged therapy for HIE, reflecting the lack of mechanistic understanding of its pathophysiology. (Macro)autophagy, a physiological intracellular process of lysosomal degradation, has been proposed to be excessively activated in excitotoxic conditions such as HIE. The present study examines whether neuronal autophagy in the thalamus of asphyxiated human newborns or P7 rats is enhanced and related to neuronal death processes. METHODS: Neuronal autophagy and cell death were evaluated in the thalamus (frequently injured in severe HIE) of both human newborns who died after severe HIE (n = 5) and P7 hypoxic-ischemic rats (Rice-Vannuci model). Autophagic (LC3, p62), lysosomal (LAMP1, cathepsins), and cell death (TUNEL, caspase-3) markers were studied by immunohistochemistry in human and rat brain sections, and by additional methods in rats (immunoblotting, histochemistry, and electron microscopy). RESULTS: Following severe perinatal asphyxia in both humans and rats, thalamic neurons displayed up to 10-fold (p < 0.001) higher numbers of autophagosomes and lysosomes, implying an enhanced autophagic flux. The highly autophagic neurons presented strong features of apoptosis. These findings were confirmed and elucidated in more detail in rats. INTERPRETATION: These results show for the first time that autophagy is enhanced in severe HIE in dying thalamic neurons of human newborns, as in rats. Experimental neuroprotective strategies targeting autophagy could thus be a promising lead to follow for the development of future therapeutic approaches.